Hematopoeisis is characterized by the successive commitment of pluripotent hematopoietic stem cells to specific cell lineages. Disruption of this orderly process is likely central to various bone marrow failure syndromes and leukemogenesis. Therefore, an understanding of the molecular mechanisms that regulate normal hematopoiesis should yield insight into these disease processes and form the basis for rational-designed therapeutic interventions. Previous studies have demonstrated a critical role for lineage-specific transcription factors in hematopoiesis. One such transcription factor is GATA-1, the founding member of a family of zinc-finger transcription factors important in development. GATA-1 is of particular importance in erythroid and megakaryocyte development, two lineages that are thought to arise from a common bipotential precursor cell. This is highlighted by the finding of GATA-1 cognate binding sites in the cis-acting regulatory sequences of virtually all erythroid and megakaryocyte-specific genes that have been studied. Mice carrying a targeted disruption of the GATA-1 locus die in mid- embryonic development with severe anemia and arrest in erythroid differentiation at a proerythroblast-like stage. Their megakaryocytes also show perturbed development with hyperproliferation and late cytoplasmic maturation arrest. Recently, a novel GATA-1 binding protein was identified through a yeast two-hybrid screen of a mouse erythroleukemia cDNA library. This protein, termed FOG for Friend of GATA-1, is a 995 amino acid protein that contains nine predicted multitype zinc fingers. It is co-expressed with GATA-1 during embryonic development. Like the GATA-1 "knock-out" mouse, targeted disruption of the FOG locus leads to death during mid embryonic development from severe anemia with arrest at the proerythroblast stage. However, in contrast to GATA-1 null megakaryocytes, there is complete failure of megakaryocytopoiesis in the FOG "knock-out" mouse. These findings suggest that FOG acts through a GATA-1-dependent pathway in erythropoiesis, but a GATA-1-independent pathway in early megakaryocytopoiesis. The goal of the proposed project is to test this model through structure-function studies of FOG. The specific aims are (1) to develop an in vitro functional assay for FOG. In preliminary studies, a FOG minus/minus hematopoietic cell line has been generated by in vitro differentiation of ES FOG minus/minus cells followed by transformation with the homeodomain gene HOX-11. These cells appear to be blocked in erythroid and megakaryocyte differentiation, but can be rescued by re-introduction of FOG cDNA. This rescue assay will be the basis of a functional in vitro assay for FOG; (2) to determine which structural subdomains of FOG, including known GATA-1 binding domains, are necessary and/or sufficient to rescue erythroid and/or megakaryocyte differentiation; and (3) identification of a putative non-GATA-1 FOG interacting protein in megakaryocytes using a yeast two-hybrid approach to screen a megakaryocyte cDNA library with FOG as a "bait."